The goal of this program project is to develop an integrated map of human chromosome (HC) 12. The program has three research projects which will be supported by two cores. The program involves six investigators, two from the Albert Einstein College of Medicine and the rest from Yale University. The overall effort will be directed by Drs. Raju Kucherlapati of AECOM and David Ward of Yale. The proposed program takes advantage of several recent developments in genome research, especially in physical mapping, and utilizes well established methodologies as well as new and innovative methods that the members of the group have developed. Dr. Daniel Cohen of CEPH will prepare a chromosome 12 specific sublibrary from his large insert yeast artificial chromosome (YAC) library. This will be accomplished by screening gridded Alu-PCR products from individual YACs with Alu-PCR products from a somatic cell hybrid, GMl0868, which contains HC12 as its sole human component. Since the global physical mapping effort at CEPH has already assembled YAC contigs encompassing a significant portion of the genome, the preparation of an HC12 library will immediately identify HC12 YAC contigs. In a second approach to generate contigs, Alu-PCR products of YAC libraries will be screened with Alu-PCR products from 140 mapped cosmids. Each of the contigs and, where appropriate, individual members of the contigs will be mapped onto chromosome 12 by fluorescence in situ hybridization (Project 1). In the later stages of the program, Project 1 will help define the orientations of the end clones in the contig, and to identify and fill gaps. In Project 2, the DNA sequences from the ends of the expected 1,000 HC12 YACs will be isolated and examined to determine if they are derived from HC12. The HC12 specific DNA segments will be sequenced and converted into sequence tagged sites (STS). These STSs and other STSs will be used to generate an STS-content map of HC12 with markers located at intervals of 100-150 kb. The precise order and distance between markers will be established by a new strategy which involves homologous recombination. This project will integrate the genetic and physical maps and will identify DNA fragments corresponding to regions where there is a paucity of genetic markers. We propose a new strategy to isolate highly polymorphic markers, use them to type the CEPH pedigrees and improve the genetic map. The goal of Project 3 is to isolate cDNAs corresponding to HC12 genes. Towards this goal, a highly comprehensive, normalized short-fragment cDNA library representing 75-80% of human genes will be constructed. DNA from individual or a small set of well-mapped non-chimeric YACs will be used to isolate cDNAs corresponding to genes present on the YACs by an affinity hybridization procedure which we have developed. A subset of each cDNA packet will be sequenced to generate an expressed sequence landmark map of HC12. These programs will be supported by a Molecular Biology core and an Informatics core.